Central Dogma Lecture 11 Transcription I

Question 1

What are the four major classes of RNA? What do they do?

Answer 1

-mRNA (messenger RNA):
-Encodes information for synthesis of proteins
-rRNA (ribosomal RNA):
-Components of ribosomes (translation machinery)
-Comprises 90% of a cell’s total RNA
-tRNA (transfer RNA)
-Used to carry amino acids to mRNA-ribosome complex during translation
-ncRNA (noncoding RNA):
-Diverse group with catalytic, structural, and/or regulatory functions
~76% of the human genome is transcribed; most products are ncRNAs

Question 2

What polymerase is used for RNA in bacteria

Answer 2

-RNA polymerase (RNAP)
-DNA-directed synthesis of RNA
-In bacteria, one type of RNA polymerase synthesizes all RNA, except primers used in DNA replication

Question 3

What makes up the RNAP holoenzyme?

Answer 3

-Core + sigma factor (𝜎)
- 𝜎 factor dissociates once RNA synthesis has been initiated

Question 4

What catalyzes the actual polymerization?

Answer 4

-The RNAP core
-Made up of α, β, β’, and ω subunits

Question 5

What do sigma factors do?

Answer 5

-Sigma factors allow RNAP to slide rapidly on DNA in search of promoter
-Holoenzyme (core + sigma) binds loosely to duplex DNA
-Different sigma factors direct transcription of different genes
-Sigma factor specifies the promoter that RNAP holoenzyme forms a stable complex with
-All sequence specific contacts are mediated by the sigma factor

Question 5

Promoter basics in bacteria

Answer 5

-RNA synthesis is initialted at specific DNA sequences called promoters
-Upstream and have negative numbers
-Usually A or G

Question 5

Where do RNAPs bind promoters in bacteria?

Answer 5

-RNAP binds tightly to promoters at 2 highly conserved regions
-Centered at -10 position
-Centered at -35 position
-Sequence in between is unimportant, but length is critical (16-19bp)

Question 6

What does transcription rate depend on in bacteria?

Answer 6

Rate of promoter forming a stable initiation complex with RNAP enzyme

Question 7

What happens with sigma factor when transcription has been initiated?

Answer 7

-Sigma is jettisoned
-Core by itself binds tightly to duplex DNA

Question 8

How are bases to be transcribed for RNA numbered?

Answer 8

-First base of RNA that is transcribed is marked as +1
-No 0

Question 9

What is the antisense strand?

Answer 9

-The template strand (or noncoding)
-Strand that is transcribed

Question 10

What is the sense strand?

Answer 10

-Coding strand (or non-template strand)
-Has same sequence as the transcribed RNA
-Promoter is on the DNA’s coding/non-template/sense strand

Question 11

Which strand is the promoter on in DNA?

Answer 11

The coding/sense strand

Question 12

Where does RNAP holoenzyme bind promoter?

Answer 12

-On the -10 and -35 regions on the same side of DNA helix as initiation site
-Binds only to one face of the promoter B-form helix

Question 13

Formation of the transcription bubble

Answer 13

-Unclear how exactly this “invasion” happens
-Binding of RNAP holoenzyme “melts” DNA from center of -10 region to just past the initiation site
-Promoter efficiency decreases with # of G-C pairs in -10 region

Question 14

What occurs with the bubble during processive transcription?

Answer 14

Eight bases of RNA transcript are hybridized with DNA in the bubble

Question 15

What is the shape of the RNAP core structure?

Answer 15

-Shape of a crab claw whose two pincers are formed by the β and β’ subunits
-In main channel (between pincers), DNA pairs with incoming NTP
-RNA exits from between β and β’ subunits (the RNA exit channel

Question 16

How does chain initiation occur?

Answer 16

-The 5’ most base of RNA is almost always A or sometimes G
-Initiating reaction is simply the coupling of two nucleotide triphosphates
-Bacterial RNAs have 5′-triphosphate group (incorporate γ-32P ATP)
-Basically initiation is started by adding two phosphates to the first base?

Question 17

Abortive initiation

Answer 17

RNAP sometimes fails to escape the promoter and releases newly synthesized RNA after ~10 nt

Question 18

What happens in a successful initiation?

Answer 18

-RNAP commences processive transcription of the template and jettisons sigma factor
-Transition from initiation complex to elongation complex
-Sigma factor can then join another core to form a new initiation complex

Question 19

What is the chemical mechanism of transcription?

Answer 19

-RNA chain elongation occurs 5’ to 3’ (same as DNA)
-Amino acid sequence “NADFDGD” forms the RNA polymerase active site
-The three D (Asp) residues are complexed to the Mg2+ ions

Question 20

Supercoiling in Transcription DNA

Answer 20

-Transcription bubble travels along DNA with RNAP in straight line
-DNA’s helical turns are pushed ahead, causing positive (left-handed) supercoiling
-DNA behind the bubble is underwound, causing negative (right-handed) supercoiling
-Topoisomerases deal with supercoiling

Question 21

What is the rate of transcription and why is it processive?

Answer 21

-20-70nt/ second
-RNAP acts as its own clamp by binding tightly to DNA-RNA complex

Question 22

How often does transcription happen?

Answer 22

-For RNAs needed in large quantities, synthesis is initiated as often as sterically possible, about once per second
-Once an RNAP has begun elongating, another can follow

Question 23

What happens when an NTP is incorporated wrong into RNA

Answer 23

-DNA-RNA hybrid helix becomes distorted

Question 24

How does RNAP fix mistakes?

Answer 24

-RNAP backtracks a couple of nucleotides
-Normally energetically disfavored because it breaks base pairing
-Protein induces RNAP active site to hydrolyze phosphodiester bond
-RNAP corrects mistakes and resumes synthesis

Question 25

What protein induces RNAP active site to hydrolyze phosphodiester bond when the wrong NTP is incorporated and what type of activity is this?

Answer 25

-GreA or GreB in E. coli
-TFIIS in eukaryotes
-This is an endonuclease activity, not exonuclease
-RNAP cleaves two nucleotides back, not final nucleotide

Question 26

Rifamycin and Rifampicin

Answer 26

-Inhibit RNAP
-Used against tuberculosis
-Bind bacterial β subunit of RNAP
-Do not bind eukaryotic RNAPs
-Block RNA chain elongation
-Single SNP mutations in rpoB (encoding β) provide resistance
-Don’t naturally have this resistance because it makes it less efficient/effective

Question 27

Intrinsic terminator site

Answer 27

-Does not require assistance
-Palindromic G-C rich segment (2-fold symmetry)
-Followed by 7-10 As on template strand

Question 28

What does the intrinsic terminator site cause the formation of?

Answer 28

-Forms a stable self-complementary hairpin (G-C)
-Followed by Us with weak base pairing to template DNA
-Mutations to these structures can eliminate chain termination

Question 29

What do the termination sites use for termination?

Answer 29

-Half use Intrinsic termination with strong hairpin
-Half use Rho factor (ρ)

Question 30

Rho-mediated termination

Answer 30

-Rho attaches to nascent RNA at its recognition sequence (rut)
-Rho translocates along ssRNA transcript 5’ to 3’ until it bumps into RNAP
-Rho pushes RNAP forward causing unwinding of RNA-DNA helix and rewinding of dsDNA

Question 31

rut sequence

Answer 31

-Rho (ρ) recognition site
-80-100 nt long
-lacks secondary structure
-G-C rich